Protein N-terminal Methylation Mechanisms and Inhibition

蛋白质 N 末端甲基化机制和抑制

• 批准号: 9978827
• 负责人: Rong Huang
• 金额: $ 37.29万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-20 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978827
• 关键词: Aging; Amines; Amino Acid Motifs; Binding; Biochemical; Biochemical Pathway; Bioinformatics; Biological; Biological Assay; Biological Process; Biology; Catalysis; Cell Extracts; Cell physiology; Cells; Cellular Stress; Cellular Stress Response; Chemical Structure; Chemicals; Chromatin; Complex; Coupled; Crystallization; DNA Damage; DNA Repair; Data; Defect; Development; Developmental Process; Disease; Enzymes; Evaluation; Exhibits; Foundations; Functional disorder; Goals; Immunoprecipitation; Kinetics; Knock-out; Knockout Mice; Lead; Link; Malignant Neoplasms; Mediating; Methylation; Methyltransferase; Mitosis; Modification; Molecular; Molecular Structure; Mutation; Mutation Analysis; N-terminal; Pathogenesis; Pathway interactions; Peptides; Pharmacology; Phenotype; Photoaffinity Labels; Physiological; Play; Premature aging syndrome; Property; Protein Methyltransferases; Proteins; Reaction; Reader; Regulation; Research; Roentgen Rays; Role; Route; Series; Specificity; Structure; Structure-Activity Relationship; Time; Western Blotting; Work; analog; cofactor; database structure; demethylation; experimental study; human disease; inhibitor/antagonist; interdisciplinary approach; knock-down; knowledge base; mouse model; mutant; novel; novel therapeutic intervention; protein function; public health relevance; small molecule inhibitor; success; tool

Protein N-terminal methyltransferases (NTMTs) are a relatively new type of protein methyltransferase that catalyze the methylation of alpha-N-terminal amines of proteins starting with an X-P-K/R motif. The alpha-N- terminal methylation plays an essential role in regulating cell mitosis, chromatin interactions, and DNA repair. Its level is increased as response of cellular stress, aging, and developmental processes. Aberrant expression of NTMTs has been observed in various human diseases. However, the biological impact of protein alpha-N- terminal methylation and the molecular basis of NTMT catalysis are poorly defined. Our long-term research goal is to elucidate the biochemical pathways mediated by NTMTs that contribute to the pathogenesis of cancer and developmental defects, and to develop potent and specific NTMT inhibitors. The objective of this research to study mechanism, regulation, and recognition of protein alpha-N-terminal methylation through exploring and applying new chemical biology approaches. Three specific aims will be pursued: Aim 1. Elucidate the molecular and structural basis for substrate and product specificity of NTMTs. We will apply mutational and crystallographic analyses to understand how NTMT1 and 2 recognize their substrates and produce specific methylated products. In addition, we propose to identify a bioorthogonal pair of mutant NTMT1 and SAM analog and use it to obtain a comprehensive profile of physiological substrates for NTMT1. We will carry out biochemical assays and cellular methylation assays to confirm the relevance of identified substrates in cells. Aim 2. Develop potent and specific inhibitors for NTMTs. We will build on our preliminary data to complete synthesis and evaluation of a series of bisubstrate analogs for NTMTs, which will be used as probes to characterize the structural and functional differences of NTMT1 and 2, to investigate NTMT inhibition in cells in comparison with the effects of NTMT1 knockdown. We will extend our crystal structure database of NTMT ternary complexes and to screen for small molecule inhibitors for NTMTs. Aim 3. Identify readers and erasers for protein alpha-N-terminal methylation. There is no information on how this N-terminal methylation is `read' by interacting molecular partners and whether it is static or dynamic (`erasable'). We propose to prepare photoaffinity labeling probes as baits to identify interacting partners of protein alpha-N-terminal methylation in cell extracts. Identified interacting partners will be validated through binding studies and immunoprecipitation experiments. Bioinformatics analysis will be done to develop hypotheses for the biological functions of these interacting partners. Taken together, we believe that this research effort has the great potential to provide a clearer understanding of mechanisms and inhibition of NTMTs, and shed lights on the biological impact of protein N-terminal methylation. Accomplishment of the proposed work will also provide new chemical tools for both basic NTMT biology research and facilitate the development of novel therapeutic approaches to target NTMT1 and NTMT1-involved pathways.

蛋白质N末端甲基转移酶(NTMTs)是一种相对较新的蛋白质甲基转移酶 催化从X-P-K/R基序开始的蛋白质的α-N端胺的甲基化。字母-N- 末端甲基化在调节细胞有丝分裂、染色质相互作用和DNA修复中起着至关重要的作用。 它的水平随着细胞应激、衰老和发育过程的反应而增加。异常表达 已经在各种人类疾病中观察到了NTMT的存在。然而，蛋白质α-N-的生物影响- 末端甲基化和NTMT催化的分子基础还不清楚。我们的长期研究 目的是阐明NTMTs介导的生化途径在糖尿病的发病机制中的作用。 癌症和发育缺陷，并开发有效和特异的NTMT抑制剂。这样做的目的是 蛋白质α-N末端甲基化的机制、调控和识别研究进展 探索和应用新的化学生物学方法。将追求三个具体目标：目标1。 阐明NTMTs底物和产物特异性的分子和结构基础。我们会申请 突变和结晶学分析以了解NTMT1和2如何识别它们的底物和 生产特定的甲基化产物。此外，我们建议鉴定一对生物正交变异体 NTMT1和SAM模拟，并使用它来获得NTMT1的生理底物的全面概况。 我们将进行生化分析和细胞甲基化分析，以确认已鉴定的 细胞中的底物。目的2.开发有效和特异的NTMTs抑制剂。我们将在我们初步的基础上 完成一系列NTMTs双取代类似物的合成和评价的数据，这些数据将用作 鉴定NTMT1和NTMT2的结构和功能差异，研究NTMT的抑制作用 与NTMT1基因敲除的效果进行比较。我们将扩展我们的晶体结构数据库 NTMT三元络合物和筛选用于NTMT的小分子抑制剂。目标3.确定读者和 蛋白质α-N末端甲基化的橡皮擦。目前还没有关于这种N末端甲基化是如何发生的信息 相互作用的分子伙伴的‘阅读’，以及它是静态的还是动态的(‘可擦除’)。我们建议准备 光亲和标记探针作为诱饵识别蛋白质α-N末端甲基化的相互作用伙伴 细胞提取液。将通过结合研究和免疫沉淀来确认已确定的相互作用伙伴 实验。将进行生物信息学分析，以开发这些生物功能的假说 互动伙伴。综上所述，我们相信这项研究工作具有巨大的潜力，可以提供 更清楚地了解非结核分枝杆菌的机制和抑制作用，并揭示其生物影响 蛋白质N末端甲基化。拟议工作的完成还将提供新的化学工具 基础NTMT生物学研究和促进靶向治疗新方法的开发 NTMT1和NTMT1参与的通路。